Parkinson's disease (PD) results from loss of dopamine (DA) neurons in substantia nigra (SN). The cause of this cell death is unknown, although there is considerable evidence that oxidative stress plays a significant role. Our research group has considerable experience with several models of PD in which oxidative stress is induced in dopaminergic cells by exposure to 6-hydroxydopamine (6-OHDA) or to DA itself. Both 6-OHDA and DA can cause the selective death of dopaminergic cells that can be blocked both by several drugs, including those that block uptake of 6-OHDA by the cells and that increase cellular antioxidant defenses. Particularly interesting is the capacity of trophic factors such as glial cell line-derived neurotrophic factor (GDNF) to attenuate the neurotoxic effects of oxidative stress. Over the past two years, we have developed an in vitro model of cell death/cell survival using the dopaminergic cell line, MN9D. Using these cells, we have shown that 6-OHDA causes cell death that is accompanied by several measurable cellular responses, that GDNF attenuates some of those responses, and that these protective effects can in turn be blocked by inhibitors of PI3 kinase or MEK. We now propose to further develop a model system to screen chemical libraries for compounds with neuroprotective activity that can be adapted to high throughput screening (HTS). We will focus on two specific aims. First, using Hoechst reagent to detect cell death and altered nuclear morphology, we will optimize our cellular model with respect to susceptibility to 6-OHDA-induced toxicity. To do so we will identify subclones of MN9D cells that exhibit a high degree of sensitivity to dopaminergic toxins. As necessary, we will also transfect the cells to further enhance their sensitivity to oxidative stress related to dopaminergic toxins. After using 6-OHDA to select sensitive subclones, we will examine additional oxidative stressors, for example DA itself and/or MPP+. Second, we will establish the optimal conditions for evaluating these cells within an HTS assay. We then will optimize the assay conditions to enhance cell attachment and thereby obtain cells amenable to robotic manipulations requiring multiple wash steps. Finally, we will identify simple fluorescent readouts of cell death for use with an automated fluorescent plate reader. We believe that approach has the potential to generate a robust, reproducible method to identify novel compounds by HTS with therapeutic potential for the treatment of PD.